Differences in cellular behavior are central to normal human development and how injury, pathogens and mutations cause dysfunction. Many aspects of differential cellular behavior can be attributed to differences in protein localization such as reduced adhesion molecules or increased secretion of enzymes. Clathrin dependent traffic between the trans-Golgi network (TGN) and endosomes plays an important role in localization of proteins important for cell migration, cell signaling and survival. The experiments in this proposal fit into a long term goal to understand how developmental programs, mutations and cell signaling act on the traffic machinery to cause differential cellular behavior. This is the first step will be understand the molecular mechanisms leading to efficient and accurate traffic at the TGN and endosomes. It is still unclear how clathrin and its many adaptor and accessory proteins combine to provide all activities required for the complicated steps involved in traffic. These steps include selecting and concentrating cargo, generating and targeting a transport carrier in vivo. Previous identification of a network of interacting clathrin adaptors that act at TGN and endosomes has opened new avenues for understanding the molecular mechanism of clathrin dependent traffic. In this proposal, molecular mechanisms leading to fidelity in membrane traffic and a signaling pathway regulating membrane traffic in yeast will be examined. In aim1, specific hypotheses will be tested about how clathrin and adaptors ensure coats assemble at the correct membrane, that events occur in the correct order and the correct proteins are transported. Hypotheses to be tested are that 1) cooperative binding and 2) competition for space in the tightly packed clathrin coat determine when an adaptor functions. A combined approach to test these hypotheses will use in vitro biochemistry, cell fractionation, assays of membrane traffic and live-cell microscopy of specific mutations in adaptor proteins. In Aim2, a newly identified regulation of clathrin adaptors in low nutrient conditions will be investigated. Hypotheses to be tested are that 1) low nutrients inhibits membrane traffic, 2) inhibition acts at the level of adaptor modification and 3) conserved signaling pathways coordinate traffic with other cellular responses to low nutrients. PUBLIC HEALTH RELEVANCE: Mutations of clathrin adaptors are observed in cancer and inherited mental retardation, and genome wide association studies links an adaptor to schizophrenia. Understanding how mutations in adaptors lead to dysfunction at the cellular level may suggest therapeutic interventions, and determine whether adaptor alleles are risk factors for cancer, schizophrenia and other diseases.